System and method for nucleic acid sequencing by polymerase synthesis

ABSTRACT

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/438,639 filed May 14, 2003, which is a divisional application of U.S.patent application Ser. No. 09/859,104, filed May 14, 2001, now U.S.Pat. No. 6,762,048, which application is a divisional application ofU.S. patent application Ser. No. 09/460,303, filed Dec. 13, 1999, nowU.S. Pat. No. 6,255,083, which application claims priority to U.S.Provisional Application Nos. 60/112,078, filed Dec. 14, 1998 and60/115,496, filed Jan. 11, 1999. The disclosures of all of the foregoingapplications are incorporated herein by reference in their entiretiesfor all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

FIELD OF THE INVENTION

This invention relates to improved methods for sequencing and genotypingnucleic acid in a single molecule configuration. The method involvessingle molecule detection of fluorescent labeled pyrophosphate [PPi]moieties released from nucleotide triphosphate [NTPs] as a polymeraseextension product is created.

BACKGROUND OF THE INVENTION

Previous non-electrophoretic sequencing and genotyping methods have nottaken advantage of the continuous detection of PPi release fromincorporated nucleotides. In the prior art of PPi sequencing(www.pyrosequencing.com), each nucleotide Adenosine-5′ phosphate “A”,Cytidine 5′-phosphate “C”, Guanosine 5′-phosphate, Uridine 5′-phosphate“U” and Thymidine 5′-phosphate “T”, is added separately to a reactionmixture containing the nucleic acid target and a polymerase. The currentnucleotide is removed before the next is added. Incorporation of anucleotide is accompanied by release of PPi from the NTP, detected by asecondary assay for PPi. A secondary assay is required because the PPimoiety of the NTP is not labeled. Only one nucleotide can be tested percycle; if all 4 NTPs were present simultaneously, the polymerizationreaction would continue uncontrolled and no sequence information couldbe obtained. Read length is limited by loss of synchronization among thetarget nucleic acid molecules in the sample.

Other non-electrophoretic methods, such as the stepwise ligation andcleavage of probes on DNA fragments attached to microbeads, requiressynchronization of the DNA molecules that inevitably decays with eachcycle.

The present method solves these problems and has advantages over othersequencing methods. Stepwise addition of nucleotides is unnecessary, asall four nucleotides are added simultaneously. Sequence information isproduced continuously as polymerases continually incorporate all fournucleotides into growing nucleic acid [NA] chains. There is no loss ofsynchronization because single molecules are observed separately.Analysis of single molecules also allows for the use of NA fragmentstaken directly from organisms. With the present method, it is no longernecessary to prepare NA for sequencing using cloning or amplificationprocesses, although NA so prepared can still be sequenced. In addition,there is the possibility of sequencing and genotyping many differentnucleic acids on a single surface simultaneously.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the chemical structure of quenching moieties connected througha linker to uracil.

FIG. 2 describes a dye linked to the gamma phosphate of a nucleotidealso bearing a quenching moiety.

FIG. 3 is an overview of a synthetic pathway for generating nucleotideswith fluorophores on the gamma phosphate and the quenching moiety on thebase.

FIG. 4 is an overview of the system for polymerase synthesis sequencingof target nucleic acid.

FIG. 5 is a schematic view of the method where a DNA polymerase isimmobilized onto a solid support and the target nucleic acid is DNA.

FIG. 6 is a schematic view of the method where a RNA polymerase isimmobilized onto a solid support and the target nucleic acid is doublestranded DNA.

SUMMARY OF THE INVENTION

This invention provides for a method of genotyping or sequencing atarget nucleic acid [NA] comprising the steps of: i. immobilizing anucleic acid polymerase or the target nucleic acid onto a solid supportin a single molecule configuration; ii. contacting the solid supportwith a solution containing: (a) a target nucleic acid where thepolymerase is immobilized or a polymerase where the target nucleic acidis immobilized; (b) a primer nucleic acid which complements a region ofthe target nucleic acid downstream of the region to be sequenced; (c)NTP where each type of base is differently labeled on the phosphateportion, where the labels provide a unique signal that is selectivelydetectable upon incorporation of NTP into the polymerase extensionproduct; iii. permitting the polymerase to sequentially extend theprimer incorporating the NTP creating a complement to the target nucleicacid; and, iv. determining extension of the primer by detecting theunique signal from the labeled NTP to genotype or to sequence the targetnucleic acid. The method may use a solution which contains at least twodifferent types of NTP.

It is preferred that the NTPs are labeled on the gamma phosphate with afluorescent label that differentially fluoresces when the gammaphosphate is cleaved away from the nucleoside. The immobilized moietiescan further comprise an array of locations each bearing a singlemolecule of polymerase or target nucleic acid. The immobilized moietycan be a polymerase positioned as an array of individual, singlemolecules onto a solid support. For genotyping the target nucleic acidmay be a single nucleotide polymorphism. In such circumstances one needonly add a solution which has a single type of NTP. For sequencing thedetection involves a sequential detection of different types of NTP toprovide the sequence of the target nucleic acid. In a preferredembodiment, the dNTP is labeled with a fluorophore on the gammaphosphate and a quencher moiety. In another preferred embodiment thesolution contacting the solid support flows past the immobilizedpolymerase or target NA. The polymerases can be DNA dependent or RNAdependent DNA polymerases or DNA dependent RNA polymerases. The NTPs canbe ribonucleotide triphosphates [rNTPs] or deoxynucleotide triphosphates[dNTPs] depending on the target nucleic acid and the polymerase in use.

This invention further includes a system for genotyping or sequencing atarget NA comprising: i. a solid support having a surface bearing animmobilized nucleic acid polymerase or the target NA in a singlemolecule configuration; ii. a solution for contacting the surfacecontaining: (a) a target NA where the polymerase is immobilized or apolymerase where the target NA is immobilized; (b) where the polymeraserequires the use of a primer nucleic acid there is added a primer whichcomplements a region of the target NA downstream of the region to besequenced; (c) a molar excess of NTP, where each type of NTP base isdifferently labeled on the phosphate portion and where the labelsprovide a unique signal that is selectively detectable uponincorporation of the NTP into the polymerase extension product. Thesystem embraces the same embodiments identified above for the methods.Where the system involves flowing solutions, the force for the flow maybe mechanically generated or electroosmotically generated usingelectrodes.

This invention further provides for a solid support having a surfacewhere the surface has a nucleic acid polymerase array attached to itwherein the members of the array consist of individual molecules ofpolymerase. The array members are optionally addressed so that thelocations are defined and comparative information between sites can begenerated and recorded by the optical reader. The solid support may haveDNA dependent DNA polymerase or a DNA dependent RNA polymerase or a RNAdependent DNA polymerase (reverse transcriptase) attached to it.Preferably the immobilized moieties are in a single moleculeconfiguration.

DEFINITIONS

“Addressable” in the context of an array refers to members of the arraylocated in discrete and defined regions.

“Array” refers to a solid support having more than one site or locationhaving either a target nucleic acid or a nucleic acid polymerase boundthereto.

“Complements a region of the target nucleic acid downstream of theregion to be sequenced” in the context of sequencing or genotypingrefers to the fact that the primers are extended in a 3′ direction by apolymerase. Therefore the primer binds to a subsequence of the target 3′(downstream) to the target sequence that is to be determined as the 3′end of the primer is extended.

“Genotyping” is a determination of allelic content of a target DNAwithout necessarily determining the sequence content of the entire DNA.It is a subset of sequencing. For example the identification of singlenucleotide polymorphisms by determination of single base differencesbetween two known forms of an allele is a form of sequencing that doesnot require all the target DNA to be sequenced.

“Immobilizing” refers to the attachment of a target nucleic acid orpolymerase to a solid support by a means that prevents its release in areaction solution. The means can be covalent bonding or ionic bonding orhydrophobic bonding.

“Optical reader” refers to a device that can detect and record lightemitted from the labeled dNTP.

“Sequencing” refers to the determination of the order and position ofbases in a Nucleic acid.

“Single molecule configuration” refers to an array of molecules on asolid support where members of the array are present as an individualmolecule located in a defined location. The members can be the same ordifferent.

“Type of base” in the context of nucleotide triphosphates [NTPs] refersto nucleotides able to be recognized as a substrate for the polymerase.Typical bases include adenine, cytosine, guanine, uracil, or thyminebases where the type refers to the subpopulation of nucleotides havingthat base within a population of NTPs bearing different bases. Otherrarer bases or analogs can be substituted such as xanthine orhypoxanthine or methylated cytosine.

DETAILED DESCRIPTION

1. Introduction.

This invention provides for a novel means to genotype and sequence anucleic acid. The method described herein uses individual fluorogenicNTP molecules that are identified one at a time as a RNA or DNAdependent polymerase incorporates their respective NTP into theextension products. The NTPs carry two adducts: a fluorescent dyeattached to the gamma phosphate and a fluorescence quencher attached toeither the base or sugar or dye. When the quencher is attached to thebase or sugar, the NTP is hydrolyzed upon incorporation into theextension product and the liberated pyrophosphate-dye moiety becomesfluorescent. The free dye now unquenched, becomes fluorescent and itsappearance is imaged at video-rate under a microscope.

A flowing stream sweeps the dye away from the parent molecule. All fourNTPS are present simultaneously. As the polymerase continues to movealong the target nucleic acid, the nucleotide sequence is read from theorder of the released dyes.

2. Sources of target nucleic acid.

The target nucleic acid is not critical and can come from a variety ofstandard sources. It can be mRNA, ribosomal RNA, genomic DNA or cDNA.When the target is from a biological source, there are a variety ofknown procedures for extracting nucleic acid and optionally amplified toa concentration convenient for genotyping or sequence work. Nucleic acidcan be obtained from any living cell of a person, animal or plant.Humans, pathogenic microbes and viruses are particularly interestingsources.

Nucleic acid amplification methods are also known. Preferably, theamplification is carried out by polymerase chain reaction (PCR) (U.S.Pat. Nos. 4,683,202. 4,683,195 and 4,889,818; Gyllenstein et al., 1988,Proc. Natl. Acad. Sci. USA 85: 7652-7656; Ochman et al., 1988, Genetics120: 621-623; Loh et al., 1989, Science 243: 217-220; Innis et al.,1990, PCR

Protocols, Academic Press, Inc., San Diego, Calif.). Other amplificationmethods known in the art can be used, including but not limited toligase chain reaction (see EP 320,308) use of Q- beta replicase, ormethods listed in Kricka et al., 1995, Molecular Probing, Blotting, andSequencing, Chap. 1 and Table IX, Academic Press, New York.

3. Immobilization

The single molecule arrays of use in this invention involve a support, abioreactive or bioadhesive layer and a bioresistant layer. The supportcan be glass, silica, plastic or any other conventionally non-reactivematerial that will not create significant noise or background for thefluorescent detection methods. The bioadhesive layer can be an ionicadsorbent material such as gold, nickel, or copper (Montemagno andBachand (1999) Constructing nanomechanical devices powered bybiomolecular motors. Nanotechnology 10: 225-231), protein-adsorbingplastics such as polystyrene (U.S. Pat. No. 5,858,801), or a covalentreactant such as a thiol group. To create a patterned array of thebioadhesive layer, an electron-sensitive polymer such as polymethylmethacrylate (PMMA) coated onto the support can be etched in any desiredpattern using an electron beam followed by development to remove thesensitized polymer. The holes in the polymer are then coated with ametal such as nickel and the polymer is removed with a solvent, leavinga pattern of metal posts on the substrate. This method of electron beamlithography provides the very high spatial resolution and small featuresize required to immobilize just one molecule at each point in thepatterned array. A second means for creating high-resolution patternedarrays is atomic force microscopy. A third means is X-ray lithography.

The biologics can be attached to the bioadhesive pattern by providing apolyhistidine tag on the biologic that binds to metal bioadhesivepatterns. Other conventional means for attachment employ homobifuctionaland heterobifunctional crosslinking reagents. Homobifunctional reagentscarry two identical functional groups, whereas heterobifunctionalreagents contain two dissimilar functional groups to link the biologicsto the bioadhesive. A vast majority of the heterobiftnctionalcross-linking agents contain a primary amine-reactive group and athiol-reactive group. Covalent crosslinking agents are selected fromreagents capable of forming disulfide (S—S), glycol (—CH(OH)—CH(OH)—),azo (—N═N—), sulfone (—S(═O₂—), ester (—C(═O)—O—), or amide (—C(═O)—N—)bridges.

A bioresist layer may be placed or superimposed upon the bioadhesivelayer either before or after attachment of the biologic to thebioadhesive layer. The bioresist layer is any material that does notbind the biologic. Examples include bovine serum albumin, gelatin,lysozyme, octoxynol, polysorbate 20 (polyethenesorbitan monolaurate) andpolyethylene oxide containing block copolymers and surfactants (U.S.Pat. No. 5,858,801). Deposition of the layers is done by conventionalmeans, including spraying, immersion and evaporative deposition(metals).

4. Labeling of NTPs

A. Attachment of a γ-Phosphate Fluorophore

The methods of the present invention involve detecting and identifyingindividual fluorogenic dNTP molecules as a polymerase incorporates theminto a single nucleic acid molecule. In certain aspects, a fluorescentdye is attached to the 65 -phosphate and a quencher is attached to thenucleobase. As such, the present invention provides a nucleotidetriphosphate (NTP) probe, comprising: a NTP having a 65 -phosphate witha fluorophore moiety attached thereto; a quencher moiety sufficientlyproximal to the fluorophore moiety to prevent fluorescence of thefluorophore moiety; wherein the fluorophore moiety exists quenched withat least about a 5 fold quenching efficiency, preferably, at least a 10fold quenching efficiency, when the 7-phosphate is attached to the NTPand unquenched when the y-phosphate is detached from the NTP.

In preferred aspect, the NTP probe is a dNTP probe having a fluorescentdye attached to the γ-phosphate moiety and a quencher attached to thenucleobase. Suitable nucleobases include, but are not limited to,adenine, guanine, cytosine, uracil, thymine, deazaadenine anddeazaguanosine. The quenched dNTPs are non-fluorescent when they-phosphate is attached to the NTP, and thereafter become fluorescentwhen the γ-phosphate is unattached to the NTP.

B. Fluorescence Quenching

In single molecule detection, high quenching efficiency is advantageousas it reduce fluorescence background, thus permitting the use of highernucleotide concentrations. Several quenching mechanisms exist (see, forexample, G. G. Guilbault, Practical Fluorescence, Marcel Dekker, NewYork, 1973). In certain instances, the quenching depends on spectraloverlap between fluorophore and quencher, and it finctions at long range(fluorescence resonance energy transfer, FRET). In other instances, thefluorophore and quencher interact between molecular orbitals and requirecontact between fluorophore and quencher e.g electron transfermechanisms. In still other instances, a ground-state complex quenchingmechanism can occur. All such quenching mechanisms are within the scopeof the present invention.

In certain aspects, the fluorophore moiety are fluorescent organic dyesderivatized for attachment to γ-phosphate directly or via a linker.Preferably, quencher moieties are also organic dyes, which may or maynot be fluorescent, depending on the particular embodiment of theinvention. For example, in one embodiment of the present invention, thefluorophore and the quencher are both fluorescent. In this embodiment, afluorescent energy transfer mechanism can be used wherein the firstfluorophore (e.g. fluorescein) is excited and emission is read from thesecond fluorophore (e.g. rhodamine). In these systems, dequenching isaccomplished by hydrolysis of the fluorophore attached to theγ-phosphate.

In another embodiment, the fluorophore and quencher function by anelectron transfer mechanism. In this aspect, a non-fluorescent quenchere.g DABCYL or dinitrophenyl (see, FIG. 1) absorbs energy from an excitedfluorophore, but does not release the energy radiatively. Thesequenchers can be referred to as chromogenic molecules.

There is a great deal of practical guidance available in the literaturefor providing an exhaustive list of fluorescent and chromogenicmolecules and their relevant optical properties (see, for example,Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2ndEdition (Academic Press, New York, 1971); Griffiths, Colour andConstitution of Organic Molecules (Academic Press, New York, 1976);Bishop, Ed., Indicators (Pergamon Press, Oxford, 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene, 1992) Pringsheim, Fluorescence and Phosphorescence (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing fluorophore and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleotide, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760.

Suitable donors and acceptors operating on the principle of fluorescenceenergy transfer (FET) include, but are not limited to,4-acetamido4′-isothiocyanatostilbene-2,2′ disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1 -sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy4′,5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinirnidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.

Preferred fluorophore-quencher pairs include, but are not limited to,xanthene dyes, including fluoresceins, and rhodamine dyes. Many suitableforms of these compounds are widely available commercially withsubstituents on their phenyl moieties which can be used as the site forbonding or as the bonding functionality for attachment to theγ-phosphate or nucleobase. Another group of fluorescent compounds arethe naphthylamines, having an amino group in the alpha or beta position.Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-toluidinyl-6-naphthalene sulfonate. Other dyes include3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

Preferably, the fluorophore/quencher pair are selected from fluoresceinand rhodamine dyes. These dyes and appropriate linking methodologies forattachment to nucleotides are described in many references. (see, Khannaet al (cited above); Marshall, Histochemical J., 7:299-303 (1975);Menchen et al., U.S. Pat. No. 5,188,934; Menchen et al., European PatentApplication 87310256.0; and U.S. Pat. No. 5,366,860, issued to Bergot etal.).

In a preferred embodiment, the quencher4-(4′-dimethylaminophenylazo)-benzoic acid (DABCYL) is used. Thequencher DABCYL (see, FIG. 1) is commonly used in fluorogenic probes todetect DNA hybridization (molecular beacons) or protease activity.DABCYL quenches fluorescence from a wide variety of dyes emittingbetween 475 nm and 805 nm, with measured efficiencies ranging from 90 to99.9% (see, S. Tyagi et al., Nat. Biotechnol. 16, 49 (1998); and G. T.Wang et al., Tetrahedron Lett. 31, 6493 (1990)). Without being bound byany particular theory, it is believed that the quenching mechanism ofDABCYL probably involves electron transfer, rather than fluorescenceresonance energy transfer, because it is wavelength-independent. In anequally preferred embodiment, the quenchers dinitrophenyl (DNP) ortrinitrophenyl (TNP) are used.

Quenching efficiency as measured in any particular experiment depends onthe purity of the dye-quencher pair (contaminating free dye or cleavedmolecules fluoresce); the electronic coupling and physical distancebetween dye and quencher (closer is usually better); and theexcited-state lifetime of the dye (the longer the time, the greater thechances for electron transfer).

In certain embodiments, certain visible and near IR dyes are known to besufficiently fluorescent and photostable to be detected as singlemolecules. In this aspect the visible dye, BODIPY R6G (525/545), and alarger dye, LI-COR's near-infrared dye, IRD-38 (780/810) can be detectedwith single-molecule sensitivity and are used to practice the presentinvention.

There are many linking moieties and methodologies for attachingfluorophore or quencher moieties to nucleotides, as exemplified by thefollowing references: Eckstein, editor, Oligonucleotides and Analogues:A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al.,Nucleic Acids Research, 15: 5305-5321 (1987) (3′ thiol group onoligonucleotide); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′ sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′ phosphoamino groupvia Aminolink™. II available from Applied Biosystems, Foster City,Calif.) Stabinsky, U.S. Pat. No. 4,739,044 (3′ aminoalkylphosphorylgroup); Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990)(attachment via phosphoramidate linkages); Sproat et al., Nucleic AcidsResearch, 15: 4837 (1987) (5′ mercapto group); Nelson et al., NucleicAcids Research, 17: 7187-7194 (1989) (3′ amino group); and the like.

With reference to FIG. 2, the preferred linkers to several bases ofvarious dNTP structures of the present invention are shown. Again,DABCYL is a wavelength-independent fluorescence quencher havingquenching efficiencies in the range required by the present methods.Better quenching permits higher dNTP concentrations and faster turnoverrates. In certain aspects, the quencher is linked to the same nucleobasesites typically employed for attaching dyes (see, FIG. 1). As thepolymerase progresses along the DNA, the quencher will remain at everyincorporated base. In certain aspects, the quencher is covalentlyattached to a dNTPs using the C5 linker shown in FIG. 1. In certainembodiments, the quencher moiety is attached to the fluorophore moietyvia a linker. In certain other embodiments, the quencher can be attachedto the sugar of the dNTPs.

In general, nucleoside labeling can be accomplished using any of a largenumber of known nucleoside labeling techniques using known linkages,linking groups, and associated complementary functionalities. Thelinkage linking the quencher moiety and nucleoside should be compatiblewith relevant polymerases and not quench the fluorescence of thefluorophore moiety.

Preferably, the quenchers are covalently linked to the 5-carbon ofpyrimidine bases and to the 7-carbon of 7-deazapurine bases. Severalsuitable base labeling procedures have been reported that can be usedwith the present invention, e.g Gibson et al., Nucleic Acids Research,15: 6455-6467 (1987); Gebeyehu et al., Nucleic Acids Research, 15:4513-4535 (1987); Haralambidis et al., Nucleic Acids Research, 15:4856-4876 (1987); Nelson et al., Nucleosides and Nucleotides, 5(3)233-241 (1986); Bergstrom, et al., JACS, 111, 374-375 (1989); U.S. Pat.Nos. 4,855,225, 5,231,191, and 5,449,767, each of which is incorporatedherein by reference. Preferably, the linkages are acetylenic amido oralkenic amido linkages, the linkage between the quencher and thenucleotide base being formed by reacting an activatedN-hydroxysuccinimide (NHS) ester of the dye with an alkynylamino- oralkenylamino-derivatized base of a nucleotide. More preferably, theresulting linkages are shown in FIG. 2.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0, and Hobbs et al.,J. Org Chem., 54: 3420 (1989), which is incorporated herein byreference. As taught therein, the alkynylamino-derivatized nucleotidesare formed by placing the appropriate halodeoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine deoxynucleosides and Cu(I) ina flask, flushing with argon to remove air, adding dry DMF, followed byaddition of an alkynylamine, triethyl-amine and Pd(0). The reactionmixture can be stirred for several hours, or until thin layerchromatography indicates consumption of the halodeoxynucleoside.

As taught in U.S. Pat. No. 5,047,519, which issued to Hobbs et al. onSep. 10, 1991, the alkynylamino linkers have the structure:

Nuc-C≡C-R¹-NR²R³

wherein R¹ is a substituted or unsubstituted diradical moiety of 1-20atoms. Nuc is a purine or pyrimidine base. R¹ can be straight-chainedalkylene, C₁-C₂₀, optionally containing within the chain double bonds,triple bonds, aryl groups or heteroatoms such as N, O or S. Theheteroatoms can be part of such functional groups as ethers, thioethers,esters, amines or amides. Preferably, R¹ is straight-chained alkylene,C₁-C₂₀; most preferably R¹ is CH₂. Substituents on R¹ can include C₁-C₆alkyl, aryl, ester, ether, amine, amide or chloro groups. R² and R³ areindependently H, alkyl, C₁-C₄, or a protecting group such as acyl,alkoxycarbonyl, a fluorophore, a quencher or sulfonyl. Preferably R² isH, and R³ is a quencher. The alkynylamino linker is preferably attachedto the 5-position of the pyrimidine nucleotides and the 7 position ofthe purine nucleotides.

In a preferred embodiment, a quencher-sulfoNHS derivative e.g.,DABCYL-sulfoNHS is prepared using DABCYL and reacting it withN-hydroxysulfosuccinimide and N,N′-dicyclohexylcarbodiimide under anitrogen atmosphere (see, FIG. 1). The DABCYL-sulfoNHS derivative isreacted with an aminoallyl-dNTP e.g. aminoallyl-dUTP, to produce theDABCYL-dUTP. Using the DABCYL-DUTP and cystamine dihydrochloride, aDABCYL-dUTP-thiol derivative can be generated. Thereafter, a NTP havinga y-phosphate fluorophore attached can be produced by reacting forexample the DABCYL-dUTP-thiol with BODIPY TR-iodoacetamide (commerciallyavailable from Molecular Probes D-6011) to produce DABCYL-dUTP-BODIPYTR.

C. Ouenching Efficiency

The present invention provides NTP molecules having a γ-phosphate with afluorophore moiety attached thereto. The fluorophore moiety existsquenched with at least about a 5 fold quenching efficiency when theγ-phosphate is attached to the NTP and is unquenched i.e., isfluorescent, when the γ-phosphate is detached from the NTP. Preferably,the fluorophore moiety exists quenched with at least about a 3 foldquenching efficiency to about 100 fold quenching efficiency. In a morepreferred embodiment, the fluorophore moiety exists quenched with atleast about a 100 fold quenching efficiency to about a 1000 foldquenching efficiency.

The quenching efficiency of the NTPs of the present invention is aroutine parameter easily determined. As will be apparent to those ofskill in the art, quenching efficiency can be measured in a fluorometeroptionally having laser excitation. Similar to the earlier discussion ofthe Stem-Volmer equation, quenching efficiency is equal to

F_(o)-F/F_(o)

wherein F_(o) is fluorescence of the NTP without quenching and F is thequenched fluorescence. Since there is no certain way to eliminate allfluorescence in a sample of quenched NTP, the unquenched measurements,F_(o), can be taken in a separate sample containing dye alone and thequenched measurements, F, can be made on the same concentration ofquenched dNTP.

The compounds of the present invention have at least 3 fold quenchingefficiency. A fully fluorescent dye has a F_(o) value of 1, whereas adye quenched by 90% has an F value of 0.100. A compound quenched by 90%,has a quenching efficiency of 0.9 or is 10 fold quenched. Therefore, forcompounds of the present invention, F is characterized as follows: 0.670≦F≦0.999, i.e., the compounds possess quenching efficiencies betweenabout 3 fold to about 1000 fold. Preferably the quenching efficiency ofa compound of the present invention is about at least 5 fold to about1000 fold, and more preferably, the quenching efficiency is about atleast 10 fold to about 1000 fold.

In the present invention, detection of pyrophosphate depends ongenerating a fluorescent signal by dequenching, or turning on, aquenched fluorescent dye in response to pyrophosphate. Efficientquenching provides a lower background fluorescence, enhancing thesignal-to-noise ratio upon dequenching by pyrophosphate. Incompletequenching results in a low level fluorescence background from each dyemolecule. Additional background fluorescence is contributed by a few ofthe dye molecules that are fully fluorescent because of accidental(i.e., pyrophosphate-independent) dequenching, for example by breakageof a bond connecting the dye to the quencher moiety. Thus, thebackground fluorescence has two components: a low-level fluorescencefrom all dye molecules, referred to herein as “distributed fluorescencebackground” and fill-strength fluorescence from a few molecules,referred to herein as “localized fluorescence background”.

In instances where a multi-labeling scheme is utilized, a wavelengthwhich approximates the mean of the various candidate labels' absorptionmaxima may be used. Alternatively, multiple excitations may beperformed, each using a wavelength corresponding to the absorptionmaximum of a specific label. Table I lists examples of various types offluorophores and their corresponding absorption maxima

TABLE I Candidate Fluorophores Absorbance/Emission Rho123 507/529 R6G528/551 BODIPY 576/589 576/589 BODIPY TR 588/616 Nile Blue 627/660BODIPY 650/665 650/665 Sulfo-IRD700 680/705 NN382 778/806Tetramethylrhodamine 550 Rodamine X 575 Cy3 TM 550 Cy5 TM 650 Cy7 TM 750

5. Miscellaneous Reaction Reagents

The polymerase selected for use in this invention is not critical.Preferred polymerases are able to tolerate labels both on the nucleobaseand on the gamma-phosphate. The polymerase should have a fidelity(incorporation accuracy) of at least 99% and a processivity (number ofnucleotides incorporated before the enzyme dissociates from the DNA) ofat least 20 nucleotides, with greater processivity preferred. Examplesinclude T7 DNA polymerase, T5 DNA polymerase, HIV reverse transcriptase,E. coli DNA pol I, T4 DNA polymerase, T7 RNA polymerase, Taq DNApolymerase and E. coli RNA polymerase. Exonuclease-defective versions ofthese polymerases are preferred.

The primers (DNA polymerase) or promoters (RNA polymerase) aresynthetically made using conventional nucleic acid synthesis technology.The complementary strands of the probes are conveniently synthesized onan automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (FosterCity, Calif.) model 392 or 394 DNA/RNA Synthesizer, using standardchemistries, such as phosphoramidite chemistry, e.g. disclosed in thefollowing references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311(1992); Molko et al., U.S. Pat. No. 4,980,460; Koster et al., U.S. Pat.No. 4,725,677; Caruthers et al., U.S. Pat. Nos. 4,415,732; 4,458,066;and 4,973,679; and the like. Alternative chemistries, e.g. resulting innon-natural backbone groups, such as phosphorothioate, phosphoramidate,and the like, may also be employed provided that the resultingoligonucleotides are compatible with the polymerase. They can be orderedcommercially from a variety of companies which specialize in customoligonucleotides such as a Operon Inc. (Alameda, Calif.).

Primers in combination with polymerases are used to sequence target DNA.Primer length is selected to provide for hybridization to complementarytemplate DNA The primers will generally be at least 10 bp in length,usually at least between 15 and 30 bp in length. Primers are designed tohybridize to known internal sites on the subject target DNA.Alternatively, the primers can bind to synthetic oligonucleotideadaptors joined to the ends of target DNA by a ligase. Similarly wherepromoters are used, they can be internal to the target DNA or ligated asadaptors to the ends.

6. Reaction Conditions

The reaction mixture for the sequencing comprises an aqueous buffermedium which is optimized for the particular polymerase. In general, thebuffer includes a source of monovalent ions, a source of divalentcations and a buffering agent. Any convenient source of monovalent ions,such as KCl, K-acetate, NH4-acetate, K-glutamate, NH₄Cl, ammoniumsulfate, and the like may be employed, where the amount of monovalention source present in the buffer will typically be present in an amountsufficient to provide for a conductivity in a range from about 500 to20,000, usually from about 1000 to 10,000, and more usually from about3,000 to 6,000 micromhos.

The divalent cation may be magnesium, managanese, zinc and the like,where the cation will typically be magnesium. Any convenient source ofmagnesium cation may be employed, including MgCl₂, Mg-acetate, and thelike. The amount of Mg ion present in the buffer may range from 0.5 to20 mM, but will preferably range from about 1 to 12mM, more preferablyfrom 2 to 10 mM and will ideally be about 5 mM.

Representative buffering agents or salts that may be present in thebuffer include Tris, Tricine, HEPES, MOPS and the like, where the amountof buffering agent will typically range from about 5 to 150 mM, usuallyfrom about 10 to 100 mM, and more usually from about 20 to 50 mM, wherein certain preferred embodiments the buffering agent will be present inan amount sufficient to provide a pH ranging from about 6.0 to 9.5,where most preferred is pH 7.6 at 25° C. Other agents which may bepresent in the buffer medium include chelating agents, such as EDTA,EGTA and the like.

7. Sample Housing

The solid support is optionally housed in a flow chamber having an inletand outlet to allow for renewal of reactants which flow past theimmobilized moieties. The flow chamber can be made of plastic or glassand should either be open or transparent in the plane viewed by themicroscope or optical reader. Electro-osmotic flow requires a fixedcharge on the solid support and a voltage gradient (current) passingbetween two electrodes placed at opposing ends of the solid support. Theflow chamber can be divided into multiple channels for separatesequencing. Examples of micro flow chambers exist. For example, Fu etal. (Nat. Biotechnol. (1999) 17:1109) describe a microfabricatedfluorescense-activated cell sorter with 3 μm×4 μm channels that utilizeselectro-osmotic flow for sorting.

8. Detection of Fluorophores

This invention requires the imaging of single molecules in a solution.There are a variety of known ways of achieving this goal. Generalreviews are available describing this technology. Reviews include Bascheet. al., eds., 1996, Single molecule optical detection, imaging, andspectroscopy, Weinheim:VCM, and Plakhotnik, et. al., Single-moleculespectroscopy, Ann. Rev. Phys, Chem. 48: 181-212. In general, the methodsinvolve detection of laser activated fluorescence using microscopeequipped with a camera. It is sometimes referred to as a high-efficiencyphoton detection system. Nie, et. al., 1994, Probing individualmolecules with confocal fluorescence microscopy, Science 266:1018-1019.The detection of single molecules involves limiting the detection to afield of view in which one has a statistical reason to believe there isonly one molecule (homogeneous assays) or to a field of view in whichthere is only one actual point of attachment (heterogeneous assays). Thesingle-molecule fluorescence detection of the present invention can bepracticed using optical setups including near-field scanning microscopy,far-field confocal microscopy, wide-field epi-illumination, and totalinternal reflection fluorescence (TIRF) microscopy. For two-dimensionalimaging fluorescence detection, the microscope is typically a totalinternal reflectance microscope. Vale et. al., 1996, Direct observationof single kinesin molecules moving along microtubules, Nature 380: 451,Xu and Yeung 1997, Direct Measurement of Single-Molecule Diffusion andPhotodecomposition in Free Solution, Science 275: 1106-1109.

Suitable photon detectors include, but are not limited to, photodiodesand intensified CCD cameras. In a preferred embodiment, an intensifiedcharge couple device (ICCD) camera is used. The use of a ICCD camera toimage individual fluorescent dye molecules in a fluid near the surfaceof the glass slide is advantageous for several reasons. With an ICCDoptical setup, it is possible to acquire a sequence of images (movies)of fluorophores. In certain aspects, each of the NTPs of the presentinvention has a unique fluorophore associated with it, as such, afour-color instrument can be used having four cameras and four exitationlasers. Thus, it is possible to use this optical setup to sequence DNA.In addition, many different DNA molecules spread on a microscope slidecan be imaged and sequenced simultaneously. Moreover, with the use ofimage analysis algorithms, it is possible to track the path of singledyes and distinguish them from fixed background fluorescence and from“accidentally dequenched” dyes moving into the field of view from anorigin upstream.

In certain aspects, the preferred geometry for ICCD detection ofsingle-molecules is total internal reflectance fluorescence (TIRF)microscopy. In TIRF, a laser beam totally reflects at a glass-waterinterface. The optical field does not end abruptly at the reflectiveinterface, but its intensity falls off exponentially with distance. Thethin “evanescent” optical field at the interface provides low backgroundand enables the detection of single molecules with signal-to-noiseratios of 12:1 at visible wavelengths (see, M. Tokunaga et al., Biochem.and Biophys. Res. Comm. 235, 47 (1997) and P. Ambrose, Cytometry, 36,244 (1999)).

The penetration of the field beyond the glass depends on the wavelengthand the laser beam angle of incidence. Deeper penetrance is obtained forlonger wavelengths and for smaller angles to the surface normal withinthe limit of a critical angle. In typical assays, fluorophores aredetected within about 200 nm from the surface which corresponds to thecontour length of about 600 base pairs of DNA. Preferably, a prism-typeTIRF geometry for single-molecule imaging as described by Xu and Yeungis used (see, X-H.N. Xu et al., Science, 281, 1650 (1998)).

Single molecule detection can be achieved using flow cytometry whereflowing samples are passed through a focused laser with a spatial filterused to define a small volume. U.S. Pat. No. 4,979,824 describes adevice for this purpose. U.S. Pat. No. 4,793,705 describes and claims indetail a detection system for identifying individual molecules in a flowtrain of the particles in a flow cell. The '705 patent further describesmethods of arranging a plurality of lasers, filters and detectors fordetecting different fluorescent nucleic acid base-specific labels. U.S.Pat. No. 4,962,037 also describes a method for detecting an orderedtrain of labeled nucleotides for obtaining DNA and RNA sequences using anuclease to cleave the bases rather than a polymerase to synthesize asdescribed herein. Single molecule detection on solid supports isdescribed in Ishikawa, et al. (1994) Single-molecule detection bylaser-induced fluorescence technique with a position-sensitivephoton-counting apparatus, Jan. J. Apple. Phys. 33:1571-1576. Ishikawadescribes a typical apparatus involving a photon-counting camera systemattached to a fluorescence microscope. Lee et al. (1994), Laser-inducedfluorescence detection of a single molecule in a capillary, Anal. Chem.,66:4142-4149 describes an apparatus for detecting single molecules in aquartz capillary tube. The selection of lasers is dependent on the labeland the quality of light required. Diode, helium neon, argon ion,argon-krypton mixed ion, and Nd:YAG lasers are useful in this invention.

This invention can be viewed as a system of components designed todetect the synthesis of a nucleic acid as PPi is released. FIG. 4provides an overview of this system which is described in detail inExample 2. FIG. 5 illustrates a typical arrangement of the polymerase(14) immobilized on the surface of the solid support (15) with a targetDNA (13) being extended by the incorporation of gamma labeled dNTPs(17). More specifically, the target DNA is hybridized to a primer thatwill become the extension product generated by the tethered DNAdependent DNA polymerase.

FIG. 6 illustrates a continuous sequences of double stranded DNAmolecules (18) using double stranded DNA dependent RNA polymerase (19)immobilized on a solid support (15) where the extension product istranscribed as a new RNA (20) from the labelled rNTPs (21). Morespecifically, a double stranded DNA template binds to the RNA polymerasethrough sequence recognition of a polymerase promoter sequence. Thesequence is read as the DNA travels through the enzyme yielding anascent RNA. After the RNA and DNA are released, the enzyme is ready tosequence the next DNA molecule passing by.

These schemes ensure that the active site remains in the evanescentlight field at the surface, so that every dye released from a quencheddNTP is illuminated. Tethering of the polymerase, rather than the targetnucleic acid (template) is convenient because it provides for acontinuous sequencing process where one immobilized enzyme sequencesmany different DNA molecules.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

Example 1 Polymerase Array

This example shows how to fabricate an array of nickel nanodots andattach a polymerase to each dot. The required equipment includes aspinner (PWM 202 E-beam resist spinner, Headway Research Inc.), anevaporator (SC4500 thermal e-gun evaporator, CVC Products Inc.), and ascanning electron microscope (Leo 982 with Nabity pattern generator, LeoElectron Microscopy Inc.).

Clean a 25 mm diameter microscope coverslip on the spinner by sprayingalternately with acetone and isopropyl alcohol (IPA) and spinning thelast IPA film until dry. Coat the coverslip in the spinner with 0.5 mlof PMMA (poly(methyl methylacrylate), MW 496 kDa, 2% in chlorobenzene),bake on a hoplate at 170 C for 10 min, coat with 0.5 ml of PMMA (MW 950kDa, 2% in methyl isobutyl ketone [MBK]), and bake again. Apply theconductive layer by evaporating 100 Angstroms of gold onto the PMMA filmin the CVC SC4500. Use the electron microscope to etch the array patterninto the PMMA film using a pattern generator on the Leo 982 as specifiedby a CAD drawing (Design CAD, 50 nm spots, 10 μm center-to-centerspacing, 200×200 dot array).

Remove the gold layer by placing the exposed coverslip in Gold Etch(15-17% sodium iodide) for 7 seconds followed by rinsing with IPA andwater. Deposit Tantalum (50 Angstroms) and Nickel (100 Angstroms) on thecoverslip in the CVC SC4500. Remove the PMMA in a 1:1 mix of acetone andmethylene chloride for 10-15 min followed by sonication for severalseconds and rinsing with IPA and water.

Attach the polymerase just before use by applying 10 ul of a 15 nMsolution of polyhistidine-tagged Klenow DNA polymerase exo⁻ (preparedusing TOPO cloning vector and ProBond Resin, Invitrogen Inc.) inphosphate-buffered saline (PBS; Harlow E., Lane D. 1988. Antibodies ALaboratory Manual. Cold Spring Harbor Laboratory ISBN 0-87969-14-2) tothe coverslip; after 20 min, wash the coverslip in PBS and useimmediately.

Example 2 Two-Color Single-Molecule Imaging Microscope

The microscope described in FIG. 4 is used to detect single moleculeevents on the surface of the coverslip obtained in Example 1. Themicroscope is fitted with a multicolor mixed-gas laser (1) which emitslight at tunable wavelengths. The laser beam is first passed through alaser line filter (10) in order to filter out undesirable wavelengths.The unfocused output passes to a fused-silica right angle prism (2),which is located directly under the cover slip (3). The sample (4)molecules sit in a buffer solution on the cover slip.

Laser light entering the prism is refracted at an angle such that thecritical angle between fused-silica and the buffer solution is exceeded.The light is thus completely reflected within the prism, giving rise toa total internal reflection (TIR) evanescent field (9) in the buffer.The area of interest is imaged using a microscope (6) with an objectivelens (5) immersed directly in the buffer. Fluorescence emission at themicroscope output passes through a multi-wavelength viewer (7), whichspatially separates images at different wavelengths. This allows thediscrimination of events occurring at different wavelengths.

The images are projected onto the array of an intensified CCD (ICCD)camera (8), from which they are digitized and stored in memory (11).Image analysis algorithms (12) are performed on the images stored inmemory. These algorithms may distinguish signal from background, trackmolecules, and perform other signal processing finctions. The memory andsignal processing may be performed off-line in a computer, or inspecialized digital signal processing (DSP) chips controlled by amicroprocessor.

Example 3 Synthesis of Dual-Labeled Nucleotides Example 3A

The synthesis of DABCYL-dUTP-BODIPY TR. (FIG. 3).

a. Preparation of DABCYL-sulfoNHS

DABCYL (108 mg, 0.4 mmole; Aldrich 25,019-8 Methyl red) was dissolved ina mixture of dry N,N-dimethylformamide (10 mL; Aldrich 22,705-6) and drypyridine (96 mg, 1.2 mmole; Aldrich 27,097-0). N-Hydroxysulfosuccinimide(260 mg, 1.2 mmole; Pierce 24510) and N,N′-dicyclohexylcarbodiimide (250mg, 1.2 mmole; Pierce 20320) were added and the mixture was stirred at50° C. for 2 hours under a nitrogen atmosphere. The reaction wasmonitored by TLC (MKC18F Reversed Phase; Whatman 4803-110; developed in0.1 M triethylammonium acetate, pH 7, 80% acetonitrile). After dilutionwith ether, the supernatant was decanted, the product was washed withether on a filter, dried, and stored dessicated at −20° C.

b. Synthesis of DABCYL-dUTP

Aminoallyl-dUTP (10 mg, 20 μmole; Sigma A 0410) was mixed withDABCYL-sulfoNHS (30 mg, 30 μmole; from step A) in 3 mL of 0.1 M sodiumcarbonate pH 8.3. The mixture was incubated in the dark for 4 hours atroom temperature and the reaction was monitored by TLC (as in step A).The DABCYL-dUTP product was purified by reversed-phase HPLC using alinear gradient from 0% to 100% of Buffer B mixed into Buffer A over 20minutes (Buffer A is 0.1 M triethylammonium acetate in water, pH 7, 4%acetonitrile; Buffer B is the same as Buffer A with 80% acetonitrile).

c. Synthesis of DABCYL-dUTP-thiol

DABCYL-dUTP (9 mg, 12 μmole; from step B) was dissolved in ImL of 0.1 MMES pH 5.7 (Sigma M 3023) and adjusted to pH 5.75. Cystaminedihydrochloride (10 mg, 44 μmol; Sigma C 8707) was dissolved in 2.5 mLof 0.1 M MES pH 5.7 and adjusted to pH 5.75. EDC (9 mg, 47 μmol; Pierce22980) was dissolved in 0.5 mL of 0.1 M MES pH 5.7 and was addedimmediately to the DABCYL-dUTP solution. After 10 minutes, thecystarnine solution was added and the pH was maintained between 5.5 and5.8 while the reaction proceeded at room temperature. After two hours,the pH was adjusted to 7.0 and the sample was stored at −20° C. Theproduct was purified by reversed-phase HPLC as in step b.

d. Synthesis of DABCYL-dUTP-BODIPY TR

DABCYL-dUTP-thiol (2.5 mg, 3 μmole; from step C) was dissolved in 5.4 mLof 5 mM TCEP (Pierce 20490), 30 mM sodium phosphate adjusted to pH 7.5.BODIPY TR-iodoacetamide (5 mg, 7.4 umol; Molecular Probes D-601 1) wasdissolved in 2.6 mL of N,N-dimethylformamide and was added to theDABCYL-dUTP-thiol solution. After standing at room temperature in thedark for 5 hours, the product was purified by reversed-phase HPLC as instep b.

e. Determination of Quenching Efficiency

The quenching efficiency of DABCYL-dUTP-BODIPY TR was determined asfollows. First, the fluorescence of a sample containing the dye BODIPYTR is measured. Second, a sample containing the same concentration ofthe nucleotide triphosphate having a y-phosphate with a fluorophoremoiety attached i.e., DABCYL-dUTP-BODIPY TR is measured. Thereafter, thequenching efficiency, which is equal to F_(o)-F/F_(o) wherein F_(o) isfluorescence of the BODIPY TR alone and F is the fluorescence ofDABCYL-dUTP-BODIPY TR is calculated. The fluorescence quenchingefficiency of DABCYL-dUT?-BODIPY TR is at least 5 fold compared to theBODIPY TR alone.

Example 3B. Synthesis of Rho-dUTP-BODIPY TR and Rho-dCTP-BODIPY TR

a. Synthesis of Rho-dUTP

Aminoallyl-dUTP (20 umole; Sigma A 0410) was mixed with5-carboxyrhodamine 6G, succinimidyl ester (30 umole; Molecular ProbesC-6127) in 3 ml of 0.1 M sodium carbonate pH 8.3, 20% DMF. The mixturewas incubated in the dark at room temperature and the reaction wasmonitored by TLC (MKC18F Reversed Phase; Whatman 4803-110; developed in0.1 M triethylammonium acetate, pH 7, 80% acetonitrile). The Rho-dUTPproduct was purified by reversed-phase HPLC using a linear gradient from0% to 100% of Buffer B mixed into Buffer A over 20 minutes (Buffer A is0.1 M triethylammonium acetate in water, pH 7, 4% acetonitrile; Buffer Bis the same as Buffer A with 80% acetonitrile).

b. Synthesis of Rho-dUTP-thiol

Rho-dUTP (12 umole; from step a) was dissolved in 1 ml of 0.1 M MES pH5.7 (Sigma M 3023) and adjusted to pH 5.75. Cystamine dihydrochloride(44 umol; Sigma C 8707) was dissolved in 2.5 ml of 0.1 M MES pH 5.7 andadjusted to pH 5.75. EDC (47 umol; Pierce 22980) was dissolved in 0.5 mlof 0.1 M MES pH 5.7 and was added immediately to the Rho-dUTP solution.After 10 minutes, the cystamine solution was added and the pH wasmaintained between 5.5 and 5.8 while the reaction proceeded at roomtemperature. After two hours, the pH was adjusted to 7.0 and the samplewas stored at −20 C. The product was purified by reversed-phase HPLC asdescribed in step a.

c. Synthesis of Rho-dUTP-BODIPY TR

Rho-dUTP-thiol (3 umole; from step b) was dissolved in 5.4 ml of 5 mMTCEP (Pierce 20490), 30 mM sodium phosphate adjusted to pH 7.5. BODIPYTR-iodoacetamide (7.4 umol; Molecular Probes D-6011) was dissolved in2.6 ml of N,N-dimethylformamide and was added to the Rho-dUTP-thiolsolution. After standing at room temperature in the dark for 5 hours,the product was purified by reversed-phase HPLC as in step a.

d. Synthesis of Rho-dCTP-BODIPY TR

Labeled dCTP was made essentially as described for dUTP, except thataminoallyl-dCTP was substituted for the dUTP. Aminoallyl-dCTP wassynthesized according to U.S. Pat. 5,476,928 except that dCTP was usedinstead of dUTP.

Example 4 Genotyping

This example illustrates a sequence-based genotyping assay on single DNAmolecules. The target is the (Delta)F508 deletion of the cystic fibrosistransmembrane conductance regulator gene (Welsh M J, et al., 1993. J.Cell Science 106S:235-239). Genomic DNA is isolated from whole blood ofCFTR-homozygous individuals (Wizard Genomic DNA Purification Kit Cat.No. A1120, Promega Inc.). Amplify a 54-nucleotide segment of the CFTRgene by PCR using the primers 5′-CACCATTAAAGAAAATATCAT (primer 1 Seq.ID. No 1) and 5′-Biotinyl-CTGTATCTATATTCATCATAG (primer 2 Seq. ID No.2).

The custom primers are obtained from a commercial source (e.g., OperonTechnologies, Alameda, Calif.). Primer 1 hybridizes to the CFTR sequenceadjacent to the (Delta)F508 deletion; the first nucleotide 3′ to primer1 is cytosine in the normal allele or thymine in the deletion mutant(see Table 2 below; from Vaughan P, McCarthy T. 1998. Nuc Acids Res 26:810-815). Amplification conditions are 200 ng of genomic DNA, 0.2 mMeach of dATP, dCTP, dGTP and dUTP, 0.2 uM of each primer, 2.0 mM MgCl2,1 unit of Taq DNA polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 9 at 25 C),0.1% Triton X-100, in a final volume of 50 ul (30 cycles of 94 C for 30sec, 52 C for 30 sec, 72 C for 15 sec; optimization of Mg concentrationand cycling parameters may be necessary, depending on the particularcharacteristics of the thermal cycler device employed).

Purify the PCR product on streptavidin-coated magnetic beads (DYNAPUREDye Terminator Removal kit, Dynal A.S.) and re-amplify for 15 cyclesunder the same conditions as before, except replace the genomic DNA with2 ul of the purified amplification product and omit primer 1 in order toproduce an excess of the single-stranded DNA product complementary toprimer 1. Remove the dNTPs from the amplified product(“template-primer”) using magnetic beads as before.

Place 10 μl of reaction mix (30 nM template-primer, 100 nM primer 1, 1nM Rho-dCTP-BTR (Example 3), 10 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 7.5 mMdithiothreitol) onto the polymerase array on the two-colorsingle-molecule imaging microscope of Example 2 and acquire a series ofimages for 20 minutes. Similarly analyze a second sample that isidentical to the first except that Rho-dCTP-BTR of the first sample isreplaced with Rho-dUTP-BTR (Example 3). Analyze the image sets forincorporation events as indicated by separation of the two coupled dyesfrom one another. Compare the number of events observed using dCTP(sample 1) and dUTP (sample 2) to determine whether the test CFTR geneis the normal or deletion allele.

TABLE 2 Normal Allele Seq Id. No. 3 CACCATTAAAGAAAATATCATCUUUGGUGUUUCCUAUGAUGAAUAUAGAU ACAGGUGGUAAUUUCUUUUAUAGUAGAAACCACAAAGGATACTACTTATATCTA TGTC Deletion MutantSeq ID. No. 4 CACCATTAAAGAAAATATCATUGGUGUUUCCUAUGAUGAAUAUAGAUACA G.GUGGUAAUUUCUUUUAUAGUAACCACAAAGGATACTACTTATATCTATGT CThe primers are underlined (primer 1 at left, primer 2 at right). Thethree deleted base pairs are indicated in bold type in the normalsequence.

Example 5 Sequencing

Sequencing is accomplished as for genotyping (Example 4), with readingof more than one nucleotide downstream of the primer.

a) Synthesis of BODIPY TR-dUTP-Rhodamine Follow the procedure of Example3a to make BODIPY TR-dUTP,

substituting BODIPY TR-X STP ester (Molecular Probes B-10003) for therhodamine of Example 3a. Derivatize the BODIPY TR-dUTP with a thiol asin Example 3b, and conjugate tetramethylrhodamine-5-iodoacetamidedihydroiodide (Molecular Probes T-6006) to the thiol as in Example 3c.

b) Sequencing

Use the same normal allele DNA template as prepared in Example 4. Place10 ul of reaction mix (30 nM template-primer, 100 nM primer 1, 1 nMBODIPY TR-dUTP-Rho (Example 5a), 1 nM Rho-dCTP-BODIPY TR (Example 3d),10 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 7.5 mM dithiothreitol) onto thepolymerase array on the two-color single-molecule imaging microscope ofExample 2 and acquire an image series. Incorporation of dUTP is seen byseparation of the two dyes, with the rhodamine being released.Incorporation of dCTP is detected by separation of the same two dyes,but with the Rhodamine being released instead of the BODIPY.

1-72. (canceled)
 73. A method for sequencing a plurality of targetnucleic acid molecules, the method comprising: subjecting the pluralityof target nucleic acid molecules to a polymerization reaction includinga plurality of types of nucleotides or nucleotide analogs to yield apopulation of growing nucleic acid strands that are complementary totheir respective target nucleic acid molecules, wherein the targetnucleic acid molecule and/or a nucleic acid polymerase is attached to asupport, where at least one of the types of nucleotides or nucleotideanalogs includes a first atomic or molecular tag bonded to a nucleotideor nucleotide analog part released during incorporation; and opticallyidentifying a time sequence of incorporation events, at least one of theincorporation events involving the incorporation of a tagged nucleotideor nucleotide analog into at least one growing nucleic acid strand inthe population, where each event involving a tagged nucleotide ornucleotide analog is characterized by a change in a detectable propertyof the first tag.
 74. A method for sequencing a plurality of targetnucleic acid molecules, the method comprising: subjecting the pluralityof target nucleic acid molecules to a polymerization reaction in thepresence of a plurality of types of nucleotides or nucleotide analogs toyield a population of growing nucleic acid strands that arecomplementary to their respective target nucleic acid molecules, whereinthe target nucleic acid molecule and/or a nucleic acid polymerase isattached to a support, where at least one of the types of nucleotides ornucleotide analogs includes a first atomic or molecular tag bonded to anucleotide or nucleotide analog part released during incorporation; andoptically identifying in real-time or near real-time a time sequence ofincorporation events, where at least one of the incorporation eventsinvolves the incorporation of a tagged nucleotide or nucleotide analoginto at least one growing nucleic acid strand in the population, whereeach event involving a tagged nucleotide or nucleotide analog ischaracterized by a change in a detectable property of the first tag. 75.The method of claims 73 or 74, wherein the polymerase is selected fromthe group consisting of a DNA polymerase, reverse transcriptase, andmixtures thereof.
 76. The method of claim 75, wherein the polymerase isa thermostable polymerase or a thermodegradable polymerase.
 77. Themethod of claim 73, wherein the target nucleic acid molecules areselected from the group consisting of double-stranded DNA,single-stranded DNA, single stranded DNA hairpins, and DNA/RNA hybrid.78. The method of claim 73, wherein the nucleotide analogs are selectedfrom the group consisting of a ribonucleotide, a deoxyribonucleotide, amodified ribonulcleotide, a modified deoxyribonucleotide, a peptidenucleotide, a modified peptide nucleotide, and a modifiedphosphate-sugar backbone nucleotide.
 79. The method of claim 73, whereinthe tagged nucleotides or nucleotide analogs types further include oneadditional tag or a plurality of additional tags.
 80. The method ofclaim 73, wherein the first tags are selected from the group consistingof chromophores, fluorescent moieties, dyes, and fluorescentnanoparticles.
 81. The method of claim 79, wherein the additional tag isor the additional tags are attached to the nucleotides or nucleotideanalogs at a base, sugar moiety, or phosphate.
 82. The method of claim73, wherein each nucleotide and nucleotide analog type includes a firsttag and where the first tags make the nucleotide or nucleotide analogtypes distinguishable during the identifying step.
 83. The method ofclaim 79, wherein three or less of the plurality of types of nucleotidesor nucleotide analogs have a different tag.
 84. The method of claim 79,wherein the different types of nucleotides or nucleotide analogs havethe same tag but are distinguished by different properties of thenucleotides or nucleotide analogs, and/or emission properties of thetags.
 85. The method of claims 73 or 74, wherein identifying is carriedout by detecting a change in a level of fluorescence energy transfer.86. The method of claims 73 or 74, wherein the identifying is performedby spectral wavelength discrimination, measurement and separation offluorescence lifetimes, fluorophore identification, backgroundsuppression or a combination thereof.
 87. The method of claims 73 or 74,wherein the plurality of target nucleic acids are attached to thesupport.
 88. The method of claims 73 or 74, wherein each target nucleicacid is hybridized to a separate oligonucleotide which is attached tothe support.
 89. The method of claim 75, wherein the DNA polymerase isattached to the support.
 90. The method of claims 73 or 74, wherein theidentifying is carried out by reducing background noise resulting fromfree nucleotides or nucleotide analogs.
 91. The method of claim 73,wherein the tag is attached to the terminal phosphate of the nucleotidesor nucleotide analogs.
 92. The method of claim 91, wherein theidentifying is effected by detecting the tags attached to thenucleotides or nucleotide analogs.
 93. The method of claim 75, whereinthe polymerase lack 3′ to 5′ exonuclease activity.